Proteins that control cell behavior can be activated (i.e. phosphorylated, undergo conformational changes) in different locations within the cell or with different kinetics to produce essentially opposing behaviors. Deciphering the spatio-temporal control of signaling is essential to understanding normal cellular homeostasis and its perturbation in many diseases. Although we have made tremendous strides in our ability to study the activity of single proteins in living cells, it remains difficult to characterize the coordination of more than one activity, critically important in rapid morphological changes and in the interaction of different signaling pathways. Biosensors based on environment-sensing dyes, which have valuable advantages over other approaches, offer an opportunity for ready multiplex imaging using equipment available on even basic live cell imaging microscopes. Biosensors based on environment sensing dyes consist of a `recognition element', a small protein fragment that binds only to the activated state of the target protein, coupled to a bright fluorescent dye that changes fluorescence when the biosensor binds its target. This design enables study of endogenous, untagged target proteins, and provides high sensitivity because bright dyes can be directly excited. Structural changes in these dyes to enhance brightness, photostability, water solubility etc. often require compromises, as structural changes affecting one property adversely affect another. We have studied the mechanisms of dye photobleaching and response to solvent polarity, and devised novel approaches to enhance water solubility. Based on this we will design here a new generation of biosensor dyes, shifting their wavelengths to permit multiplex imaging, while maintaining the photophysical features that confer advantages on dye-based biosensors. Using the new dyes, we will build `multiplexing biosensors'to quantify activation of Cdc42 or Src simultaneously with activation of either RhoA or Rac1. These new biosensors will be used to characterize the spatio-temporal coordination of Src and Rho family activation as they generate cytoskeletal changes during macropinocytosis and transendothelial migration. This proposal will develop new methods to study the cellular `circuitry'that determines how a cell responds to its environment. Such circuits consist of complex networks of interacting proteins which can be activated in different positions within a cell to produce different cell behaviors. It is currently difficult to study the activation of more than one such protein in the same cell, especially for rapid events. The new technique enables us to better understand how circuit components interact by enabling visualization of multiple circuit components, even for rapid activation events. After the technique is developed, it will be applied to study cell engulfment of other bodies, a ubiquitous response that plays an important role in many diseases (i.e. immune cells engulf invaders, white blood cells are engulfed by blood vessel walls as they pass through them during inflammation, and cells move similarly across the blood brain barrier). Engulfment requires precise orchestration of protein interactions in time and space, an ideal challenge for the new tools.